Fluorescent dyes have many uses and are known to be particularly suitable for biological applications in which the high detectability of fluorescence is desirable. By binding to a specific biological ingredient in a sample, a fluorescent dye can be used to indicate the presence or the quantity of the specific ingredient in a sample. A variety of fluorescent dyes are available for specific fluorescent staining and quantitation of DNA and RNA, and other applications involving nucleic acids.
Unsymmetrical cyanine dyes were described long before much was known about DNA, by Brooker, et al., J. AM. CHEM. SOC. 64, 199 (1942). These dyes have since been found to be useful in fluorescent staining of DNA and RNA. The nondimeric unsymmetric cyanine dye sold under the tradename Thiazole Orange has particular advantages in the quantitative analysis of immature blood cells or reticulocytes (U.S. Pat. No.. 4,883,867 to Lee, et al. (1989) ('867 patent) and U.S. Pat. No. 4,957,870 to Lee, et al. (1990) ('870 patent); Lee, et al., Thiazole Orange: A New Dye for Reticulocyte Analysis, CYTOMETRY 7, 508 (1986)]. As indicated in the '867 and '870 patents to Lee, et al., the dye used for reticulocyte analysis must be able to penetrate the cell membrane.
The inventors have discovered that a composition that includes two suitably connected unsymmetrical cyanine dye units, i.e. a covalently bonded cyanine dye dimer in which one of the dye units contains a quaternized pyridinium moiety, is a polar compound that is unable to readily penetrate cell membranes. Nevertheless, the composition discovered by inventors is highly useful as a stain for nucleic acids because it is sensitive to even small fragments of nucleic acid polymers not contained inside living cells, e.g. in cell extracts, as well as to nucleic acids in permeabilized and/or dead cells. In addition, the novel dimers have a much higher affinity for nucleic acid than do compounds such as thiazole orange that are not dimers, lower fluorescence background for the unbound probe than known dimeric nuclear stains such as ethidium homodimer, spectra that can be distinguished from those of most other nuclear stains and good fluorescence quantum yields. These dimers are neither anticipated nor obvious in view of the unsymmetrical cyanine Thiazole Orange or related cyanine compounds containing pyridinium moieties described in the '867 patent or by Lee. et al., (1986) that are monomers.
Copending application DIMERS OF UNSYMMETRICAL CYANINE DYES (Ser. No. 07/761,177 filed 9/16/91) now abandoned, published on Apr. 1, 1993 as Int. Publ. No. WO 93106482, incorporated herein by reference, describes dimeric cyanine dyes in which one resonance structure of the unsymmetrical cyanine is quinolinium rather than pyridinium. The spectral properties of pyridinium derivatives are shifted to shorter wavelengths than those of the comparable dimers of quinolinium derivatives described in the co-pending application. Although the dimers containing pyridinium moieties also have a lesser binding affinity than the comparable quinolinium derivatives, the binding affinity of the pyridinium derivatives is considerably increased over related monomer dyes. In addition, the pyridinium derivatives unexpectedly have a faster rate of binding than the quinolinium derivatives.
Other dimer compounds that are known to bind to nucleic acids include variants of ethidium homodimer, acridine homodimers, acridine-ethidium heterodimer, and 7-hydropyridocarbazoles, see, e.g., Rye, et al., NUCLEIC ACIDS RESEARCH 19(2), 327 (1990); Haugland, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS Set 31 (1992) (incorporated by reference). Although the Rye, et al. reference mentions characteristics that influence the affinity and mode of binding dimers to DNA, the reference does not describe or anticipate the advantages of the compounds used in this invention. In particular the 1990 Rye reference does not identify the unanticipated advantage of some of the subject dyes of being bound to nucleic acids with sufficiently high affinity that they co-electrophorese with the nucleic acid and undergo only minimal transfer between different nucleic acid helices as described by Rye, et al., NUCLEIC ACIDS RES. 20, 2803 (1992). Although the binding affinity as well as the fluorescent enhancement of the pyridinium derivatives are generally less than those of the comparable quinolinium derivatives, the pyridinium derivatives have nevertheless also been found to be useful for pre-staining of electrophoretic gels and have the unexpected advantage of faster uptake of dyes.
Other unsymmetrical cyanine dye compounds with increased binding affinity that are impermeant to cells are described in co-pending U.S. patent application Ser. No. 07/833,006 now U.S. Pat. No. 5,321,130 of the same inventors. These UNSYMMETRICAL CYANINE DYES WITH CATIONIC SIDE CHAIN are also described in part in Asymmetric Cyanine Dyes for Fluorescent Staining and Quantification of Nucleic Acids, presented at the 1992 Biophysical Society/ASBMB joint meeting and abstracted at BIOPHYS. J. 61, A314 (1992). While the cationic side chain increases the binding affinity of the monomer dyes, the binding affinity is generally orders of magnitude lower than that of the related dimers.
The novel cyanine dimers described herein are not only different in structure from other dimer compounds, but differ in spectral properties, binding affinities, and binding kinetics as well. There is a need for a full spectrum of nucleic acid dyes to take advantage of different laser instrumentation and for use in multi-color applications. Furthermore, there is a need for shorter wavelength dyes for nucleic acids that have greater detectability than existing dyes.